Generation of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Astrocytes for Amyotrophic Lateral Sclerosis and Other Neurodegenerative Disease Studies

Astrocytes are increasingly recognized for their important role in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS, astrocytes shift from their primary function of providing neuronal homeostatic support towards a reactive and toxic role, which overall contributes to neuronal toxicity and cell death. Currently, our knowledge on these processes is incomplete, and time-efficient and reproducible model systems in a human context are therefore required to understand and therapeutically modulate the toxic astrocytic response for future treatment options. Here, we present an efficient and straightforward protocol to generate human induced pluripotent stem cell (hiPSC)-derived astrocytes implementing a differentiation scheme based on small molecules. Through an initial 25 days, hiPSCs are differentiated into astrocytes, which are matured for 4+ weeks. The hiPSC-derived astrocytes can be cryopreserved at every passage during differentiation and maturation. This provides convenient pauses in the protocol as well as cell banking opportunities, thereby limiting the need to continuously start from hiPSCs. The protocol has already proven valuable in ALS research but can be adapted to any desired research field where astrocytes are of interest. Key features • This protocol requires preexisting experience in hiPSC culturing for a successful outcome. • The protocol relies on a small molecule differentiation scheme and an easy-to-follow methodology, which can be paused at several time points. • The protocol generates >50 × 106 astrocytes per differentiation, which can be cryopreserved at every passage, ensuring a large-scale experimental output.

This protocol is used in: Mol.Neurodegener.(2023), DOI: 10.1186/s13024-022-00591-3 Astrocytes are increasingly recognized for their important role in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS).In ALS, astrocytes shift from their primary function of providing neuronal homeostatic support towards a reactive and toxic role, which overall contributes to neuronal toxicity and cell death.Currently, our knowledge on these processes is incomplete, and time-efficient and reproducible model systems in a human context are therefore required to understand and therapeutically modulate the toxic astrocytic response for future treatment options.Here, we present an efficient and straightforward protocol to generate human induced pluripotent stem cell (hiPSC)-derived astrocytes implementing a differentiation scheme based on small molecules.Through an initial 25 days, hiPSCs are differentiated into astrocytes, which are matured for 4+ weeks.The hiPSC-derived astrocytes can be cryopreserved at every passage during differentiation and maturation.This provides convenient pauses in the protocol as well as cell banking opportunities, thereby limiting the need to continuously start from hiPSCs.The protocol has already proven valuable in ALS research but can be adapted to any desired research field where astrocytes are of interest.

Graphical overview Background
Neurodegenerative diseases affect millions of people worldwide and as the average population age increases, there is a corresponding rise in the number of patients.Amyotrophic lateral sclerosis (ALS) is one of these neurodegenerative diseases.ALS, the most prevalent motor neuron disorder among adults, affects approximately 2 out of 100,000 individuals across a wide age range, encompassing cases from teenagers to the elderly [1].Ten percent of cases are caused by inherited familial mutations, while 90% have no family history and are therefore classified as sporadic [2].Hallmarks of ALS include toxic protein aggregations, axonal transport impairments, DNA damage, and glial reactivity, leading to extensive motor neuron death [3][4][5][6][7].This causes muscle atrophy, paralysis, and death of patients typically within 2-5 years after symptom onset, and currently, there is no cure [1].As with many other neurodegenerative diseases, the focus has been on unraveling the underlying disease mechanisms behind the apparent (motor)neuronal cell death; however, the widespread glial reactivity has recently resulted in a shift from the neurocentric perspective towards increased appreciation of the role of glial cells.Astrocytes are shown to be key players in neurodegeneration [8].As one of the most abundant glial cell types in the central nervous system, astrocytes govern the support and homeostatic maintenance of neurons and their surroundings [9].Under physiological conditions, astrocytes have many functions including neurotransmitter modulation, nutritional distribution, ion, pH, and water homeostasis, blood-brain barrier regulation, and trophic support [9,10].However, in ALS and other neurodegenerative diseases, astrocytes lose these supportive characteristics and take on a more toxic reactive role [10].Considerable knowledge about the pathophysiology of ALS involving astrocytes has been gained through the use of animal models.However, it is important to acknowledge that, like all models, they come with their inherent limitations [10].Animal models often rely on overexpression of human mutant genes, which despite showing various disease-relevant mechanisms, often fail to translate to a human context [11].Importantly, overexpression models also exclude the large and important group of sporadic patients.Furthermore, human astrocytes exhibit larger sizes, more intricate branching structures, and a greater extent of synapse interactions compared to their rodent counterparts [12][13][14].As a result, the field of animal research necessitates reinforcement from human in vitro models, and human-induced pluripotent stem cells (hiPSCs) present as highly promising candidates.With their ability for self-renewal and indefinite proliferation, as well as their possibility to generate any cell type, they hold significant potential.Several protocols for generating hiPSC-derived astrocytes exist, but many of the protocols are complex and require long timelines to reach the state of full astrocyte differentiation [15][16][17][18][19][20][21].Our protocol is based on a 25-day-long differentiation followed by a 4+ week maturation.The differentiation is based on a dual inhibition of SMAD signaling pathway with the introduction of 3D culturing [22] to generate neural progenitor cells (NPCs) and a modified astrocyte differentiation protocol from Shaltouki et al. [23] to generate astrocytes [24,25].After four weeks of maturation, > 95% of the hiPSC-derived astrocyte population is positive for

E8 Flex medium
Thaw the frozen Essential 8 c.If needed, use 1 mL/well of fresh E8 Flex medium to gently flush around the borders of the well to collect remaining cells. 5. Incubate the conical tube at 37 °C with 5% CO2 for 15 min to allow the clusters to sediment.6.After incubation, remove supernatant.7. Carefully dissolve cell pellet in 10 mL of room-temperature NIM (see Recipe 4) by pipetting up and down a few times and transfer the cell suspension to a T25 non-adherent flask.Critical: Do not pipette up and down too much to sustain cell clumps.Note: Mark as passage 0 (P0).8. Check the cell density under a light microscope and incubate the flask at 37 °C with 5% CO2. 9. Perform a medium change with 10 mL/flask of room-temperature NIM on day 1 (d1), d2, and d4.
When changing medium, place the T25 non-adherent flask in an upright position in a 25 or 50 mL sterile reservoir in the incubator and allow cells to sediment for 5-10 min (Figure 1A).Carefully transfer the flask in its upright position into the biological safety cabinet and remove approximately 9 mL of spent medium to allow the cells to remain covered in a small volume of medium.Add 10 mL of fresh room-temperature NIM per flask.Notes: a. Three to four days after the start of induction, embryoid bodies (EBs) are clearly visible (Figure 1B).EBs appear irregular around their borders for the first few days but take on a rounder form during the induction phase.b.A cell shaker is not required during neural induction.If the EBs begin to adhere to each other, perform a gentle pipetting during medium changes.See Troubleshooting if EBs attach to the bottom of the flask.3.After 30 min, remove spent Geltrex and add 1 mL/well of NMM + 5 µL/mL RevitaCell TM supplement.
Incubate the plates at 37 °C with 5% CO2 to be ready for use.4. Remove spent NMM, wash cells once with DPBS, and incubate with 1 mL/well of room-temperature accutase for 4 min at 37 °C with 5% CO2. 5.After the incubation, gently tap the side of the plate to check if cells readily detach.
Note: If cells do not detach easily, incubate for one more minute and repeat step C5. 6. Add 1 mL/well of 37 °C NMM to inactivate the accutase.

Figure 1 .
Figure 1.Cell morphology during the astrocyte differentiation protocol.A. Image illustrating the use of a sterile reservoir for flask support to allow embryoid body (EB) sedimentation during medium changes.B. Brightfield images of EBs, cell morphologies, and optimal confluence during the astrocyte differentiation protocol.Scale bar: 200 µm.d+ refers to days of astrocyte maturation.The use of patient fibroblasts for the generation of hiPSCs was approved by the ethics committee of University Hospital Leuven (number S50354 and S63792).

7 .
Gently flush along the sides of the well to detach remaining NPCs and transfer cell suspension to a 15 mL conical tube.Notes: a.If not all cells detach after 5 min incubation with accutase, use a cell scraper to collect remaining cells.b.As it is not recommended to centrifuge with volumes below 3 mL for 15 mL conical tubes, increase the volume above 3 mL by adding more NMM if needed.8. Centrifuge at 145× g for 4 min at room temperature.9. Remove supernatant and continue with step C10 for passaging or step C11 for cryopreservation.10.For passaging: a. Add 1 mL/well of 37 °C NMM + 5 µL/mL RevitaCell TM supplement and carefully pipette up and down to dissolve the cell pellet.b.Transfer 1 mL/well of cell suspension to the prepared Geltrex-coated 6-well plates with 1 mL/well of NMM + 5 µL/mL RevitaCell TM supplement, so that each well contains 2 mL of NMM + 5 µL/mL RevitaCell TM supplement.c.To evenly distribute the NPCs, carefully rock the plate from side to side.d.Examine cell distribution under the light microscope and incubate the plate at 37 °C with 5% CO2.e.The following day, perform a medium change with 2 mL/well of 37 °C NMM followed by every second day until d16.11.For cryopreservation: a. Add 1 mL/vial of room-temperature freezing medium (90% FBS and 10% DMSO) and carefully pipette up and down to dissolve the cell pellet.b.Transfer 1 mL of cell suspension to prelabeled cryovials, transfer vials to a Mr. Frosty with isopropanol, and incubate at -80 °C overnight.c.The following day, transfer vials to liquid N2 storage for long-term cryopreservation.
TMFlex supplement from the Essential 8 TM Flex medium kit at room temperature for approximately 1 h or at 2-8 °C overnight.Protect the supplement from light, as it is light sensitive.Mix the thawed supplement by gently inverting the vial a couple of times and then aseptically transfer the entire contents of the Essential 8 TM Flex supplement to the bottle of Essential 8 TM Flex basal medium.Swirl the bottle to mix.E8 Flex medium can be stored at 2-8 °C for up to two weeks.Strep solution and add SB431542 and LDN193189 fresh on the day of use.Filter sterilize and bring the NIM solution to room temperature before use.Prepare > 200 mL of bulk solution of basic medium (DMEM/F12, neurobasal medium, Pen/Strep, B-27 minus vitamin A, N-2, and L-Glutamine) and filter sterilize.Basic medium can be stored at 2-8 °C for four weeks.To prepare NMM, make an aliquot of the required volume for the day of basic medium solution and add SB431542, LDN193189, FGF, and EGF fresh on the day of use.Filter sterilize and bring the NMM solution to 37 °C before use.Plate hiPSCs on Geltrex-coated 6-well plates (Table1) in 2 mL/well of E8 Flex medium and expand according to standard protocol.See Recipes 1 and 2.Note: The use of Geltrex can be replaced by Matrigel during the entire protocol.2. After a minimum of seven days as iPSCs, cell lines are prepared for neural induction when reaching 70%-90% confluence (day 0).Remove spent E8 Flex medium, wash cells once with 1 mL/well of DPBS, and incubate with 1 mL/well of room-temperature collagenase type IV (1×) for 10-20 min at 37 °C with 5% CO2 to dissociate the colonies.See Recipe 3.After incubation, remove the spent collagenase and add 1 mL/well of room-temperature E8 Flex medium, gently scrape the loosened colonies with a cell scraper, and use a P1000 pipette to transfer the cell suspension to individual 15 mL conical tubes.
2. Geltrex coatingIt is important to keep all components at ≤ 2-8 °C during the preparation of the Geltrex coating to prevent premature solidification.To ensure this, first prepare 24.75 mL of 2-8 °C DMEM/F-12 in a 50 mL conical tube and then collect the Geltrex aliquot from -20 °C.Transfer approximately 0.5 mL of 2-8 °C DMEM/F-12 from the prepared 50 mL conical tube to the Geltrex aliquot, pipette up and down to dissolve the frozen aliquot, and transfer approximately 0.75 mL of the solution back to the 50 mL conical tube.Repeat the process a few times to transfer the entire content of the Geltrex aliquot to the 50 mL conical tube.Mix well.Geltrex coating can be stored at 2-8 °C for one week.4.Neural induction medium (NIM): d0-d6Prepare ~500 mL of bulk solution of E8 Flex medium and Pen/Strep and filter sterilize.E8 Flex + Pen/strep can be stored at 2-8 °C for two weeks.To prepare NIM, make an aliquot of the required volume for the day of E8 Flex + Pen/6.Astrocyte differentiation medium (ADM): d16-d25Prepare >200 mL of bulk solution of basic medium (neurobasal medium, Pen/Strep, N-2, NEAA, and L-Ascorbic acid) and filter sterilize.Basic medium can be stored at 2-8 °C for four weeks.To prepare ADM, make an aliquot of the required volume for the day of basic medium solution and add FGF, IGF, A, and H fresh on the day of use.Filter sterilize and bring the ADM solution to 37 °C before use.Cite as: Stoklund Dittlau, K. et al. (2024).Generation of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Astrocytes for Amyotrophic Lateral Sclerosis and Other Neurodegenerative Disease Studies.Bio-protocol 14(4): e4936.DOI: 10.21769/BioProtoc.4936.6 Published: Feb 20, 2024 Note: When ready, iPSC colonies will lift and curl up around the borders.Larger colonies might require longer incubation time.After 10 min incubation, check under light microscope every 5 min.Maximum collagenase type IV (1×) incubation time: 60 min.4. Notes: a. Use one 15 mL conical tube per 6-well plate.b.Avoid excess pipetting to sustain clumps of colonies.